Pharmacological Characterization of Purified Recombinant mTOR FRB-Kinase Domain Using Fluorescence-Based Assays

Abstract

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in nutrient sensing and cell growth and is a validated target for oncology and immunosuppression. Two modes of direct small-molecule inhibition of mTOR activity are known: targeting of the kinase active site and a unique mode in which the small molecule rapamycin, in complex with FKBP12 (the 12-kDa FK506 binding protein), binds to the FRB (FKBP12/rapamycin binding) domain of mTOR and inhibits kinase activity through a poorly defined mechanism. To facilitate the study of these processes, the authors have expressed and purified a truncated version of mTOR that contains the FRB and kinase domains and have developed homogeneous fluorescence-based assays to study mTOR activity. They demonstrate the utility of these assays in studies of active site-directed and FRB domain-directed mTOR inhibition. The results suggest that these assays can replace traditional radiometric or Western blot-based assays.

Multiplexing Terbium- and Europium-Based TR-FRET Readouts to Increase Kinase Assay Capacity

“…2D). 7 However, this signal change was negligible at the concentration of mTOR to be used in the assay (360 ng/mL). Interestingly, when PIK3C3 was titrated into the assay system, there was a slight decrease in the Tb-dependent signal at high concentrations of PIK3C3 (Fig.…”

“…To develop a multiplexed assay to measure activity of mTOR and PIK3C3, we combined a traditional TR-FRETbased activity assay for mTOR (detection of acceptor-labeled product with donor-labeled, phosphospecific antibody) 7 with an assay for PIK3C3 that measures ADP formation through a competitive displacement of an ADP-derived tracer molecule from a Eu-labeled anti-ADP antibody. We reasoned that because the mTOR assay is expected to produce only nM amounts of ADP (because only nM amounts of phosphorylated product are necessary to saturate the 2 nM of anti-p-4E-BP1 antibody that is used), the amount of ADP formed in the mTOR catalyzed reaction should not affect the signal of the PIK3C3 catalyzed reaction, which would be optimized to produce larger amounts of ADP necessary to displace the tracer from the antibody.…”

“…6,7 These assays use either a fluoresceinlabeled peptide substrate or a green fluorescent protein (GFP) fusion of a protein substrate that is paired with a Tb-labeled phosphorylation site-specific antibody that recognizes and binds to the phosphorylated substrate. Following phosphorylation of the substrate by a kinase, the Tb-labeled phosphorylation site-specific antibody (the FRET donor) brings the Tb and the fluorescein (or GFP) labels into proximity, resulting in an increase in FRET.…”

“…mTOR activity was measured using GFP-4E-BP1 and a Tb-labeled antibody that specifically recognizes phosphorylation of this substrate. 7 To measure PIK3C3 activity, we used the Adapta ® Universal Kinase assay format, which measures kinase-mediated adenosine diphosphate (ADP) formation by monitoring displacement of an Alexa Fluor ® 647-labeled ADP analog from a Eu-labeled anti-ADP antibody. In this format, displacement of the Alexa Fluor ® 647-labeled ADP analog is detected by a decrease in TR-FRET signal.…”